Adoptive cell transfer (ACT) of antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies. It is known that less-differentiated, central-memory-like (termed highly avid) CTLs are the optimal population for ACT-based immunotherapy. However, ACT with such CTLs is often not feasible due to difficulties in generating these CTLs reactive with the targeted Ag from patients. Finding a new mechanism to generate highly avid CTLs is critical for the successful ACT-based cancer therapy. The long-term goal is to elicit efficient immune responses to tumor Ags and facilitate the development and application of vaccines and immunotherapy that can be used to successfully prevent and treat cancer in humans. The objective in this application is to make a significant contribution to this goal by developing a novel approach to generate highly avid CTLs derived from induced pluripotent stem (iPS) cells, and determining the therapeutic potential of iPS cell-derived CTLs in the treatment of cancer. The central hypothesis is that iPS cells that are transduced with genes of Ag-specific T cell receptor (TCR) and stimulated with in vitro Notch signaling are capable of differentiating into large numbers of highly avid Ag-specific CTLs. The rationale for the proposed research is that, once it is known how to generate highly avid Ag-specific CTLs derived from iPS cells, there is potential to elicit efficient anti-tumor immunity by ACT of iPS-derived CTLs, resulting in new and innovative approaches to the prevention and treatment of cancer. Guided by preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Generate and characterize highly avid Ovalbumin (OVA)-specific CTLs derived from murine iPS cells; and 2) Generate and characterize highly avid melanoma-specific CTLs derived from human iPS cells. Under the first aim, an already proved in vitro and in vivo approach, which has been established as feasible in the applicant' hand, will be used to generate highly avid OVA-specific CTLs derived from murine iPS cells. Under the second aim, a similar approach as described in the first aim, will be used to generate highly avid melanoma-specific CTLs derived from human iPS cells for ACT-based cancer immunotherapy. The research proposed in this application is innovative, because it will develop a novel approach to generate highly avid Ag-specific CTLs derived from iPS cells. This unique approach can be used for the generation of highly avid tumor-specific CTLs for the treatment of cancer, and will ultimately allow the development of new treatments of immunotherapy or novel vaccines. The proposed research is significant, because (i) the iPS cell-derived immune cells are highly likely to be used in preventive and therapeutic interventions for cancer in addition to fundamentally advancing the fields of T cell biology; (ii) the research described in this application will broaden our expertise regarding the use of human and animal stem cells; (iii) these studies will provide a solid foundation for further development of highly avid immune cells derived from iPS cells, and ultimately for generating patient- and/or cancer-specific therapies.